Anatomical and molecular properties of long descending propriospinal neurons in mice

Long descending propriospinal neurons (LDPNs) are interneurons that form direct connections between cervical and lumbar spinal circuits. LDPNs are involved in interlimb coordination and are important mediators of functional recovery after spinal cord injury (SCI). Much of what we know about LDPNs comes from a range of species, however, the increased use of transgenic mouse lines to better define neuronal populations calls for a more complete characterisation of LDPNs in mice. In this study, we examined the cell body location, inhibitory neurotransmitter phenotype, developmental provenance, morphology and synaptic inputs of mouse LDPNs throughout the cervical and upper thoracic spinal cord. LDPNs were retrogradely labelled from the lumbar spinal cord to map cell body locations throughout the cervical and upper thoracic segments. Ipsilateral LDPNs were distributed throughout the dorsal, intermediate and ventral grey matter as well as the lateral spinal nucleus and lateral cervical nucleus. In contrast, contralateral LDPNs were more densely concentrated in the ventromedial grey matter. Retrograde labelling in GlyT2GFP and GAD67GFP mice showed the majority of inhibitory LDPNs project either ipsilaterally or adjacent to the midline. Additionally, we used several transgenic mouse lines to define the developmental provenance of LDPNs and found that V2b positive neurons form a subset of ipsilaterally projecting LDPNs. Finally, a population of Neurobiotin (NB) labelled LDPNs were assessed in detail to examine morphology and plot the spatial distribution of contacts from a variety of neurochemically distinct axon terminals. These results provide important baseline data in mice for future work on their role in locomotion and recovery from SCI

Mrgprd Enhances Excitability in Specific Populations of Cutaneous Murine Polymodal Nociceptors

The Mas-related G protein-coupled receptor D (Mrgprd) is selectively expressed in nonpeptidergic nociceptors that innervate the outer layers of mammalian skin. The function of Mrgprd in nociceptive neurons and the physiologically relevant somatosensory stimuli that activate Mrgprd^-expressing (Mrgprd^+) neurons are currently unknown. To address these issues, we studied three Mrgprd knock-in mouse lines using an ex vivo somatosensory preparation to examine the role of the Mrgprd receptor and Mrgprd+ afferents in cutaneous somatosensation. In mouse hairy skin, Mrgprd, as marked by expression of green fluorescent protein reporters, was expressed predominantly in the population of nonpeptidergic, TRPV1-negative, C-polymodal nociceptors. In mice lacking Mrgprd, this population of nociceptors exhibited decreased sensitivity to cold, heat, and mechanical stimuli. Additionally, in vitro patch-clamp studies were performed on cultured dorsal root ganglion neurons from Mrgprd^(–/–) and Mrgprd^(+/–) mice. These studies revealed a higher rheobase in neurons from Mrgprd^(–/–) mice than from Mrgprd^(+/–) mice. Furthermore, the application of the Mrgprd ligand β-alanine significantly reduced the rheobase and increased the firing rate in neurons from Mrgprd^(+/–) mice but was without effect in neurons from Mrgprd^(–/–) mice. Our results demonstrate that Mrgprd influences the excitability of polymodal nonpeptidergic nociceptors to mechanical and thermal stimuli

Sensing of Substrate Vibrations in the Adult Cicada Okanagana rimosa (Hemiptera: Cicadidae)

Detection of substrate vibrations is an evolutionarily old sensory modality and is important for predator detection as well as for intraspecific communication. In insects, substrate vibrations are detected mainly by scolopidial (chordotonal) sense organs found at different sites in the legs. Among these sense organs, the tibial subgenual organ (SGO) is one of the most sensitive sensors. The neuroanatomy and physiology of vibratory sense organs of cicadas is not well known. Here, we investigated the leg nerve by neuronal tracing and summed nerve recordings. Tracing with Neurobiotin revealed that the cicada Okanagana rimosa (Say) (Hemiptera: Cicadidae) has a femoral chordotonal organ with about 20 sensory cells and a tibial SGO with two sensory cells. Recordings from the leg nerve show that the vibrational response is broadly tuned with a threshold of about 1 m/s2 and a minimum latency of about 6 ms. The vibratory sense of cicadas might be used in predator avoidance and intraspecific communication, although no tuning to the peak frequency of the calling song (9 kHz) could be found

Encoding of Tactile Stimuli by Mechanoreceptors and Interneurons of the Medicinal Leech

For many animals processing of tactile information is a crucial task in behavioral contexts like exploration, foraging, and stimulus avoidance. The leech, having infrequent access to food, developed an energy efficient reaction to tactile stimuli, avoiding unnecessary muscle movements: The local bend behavior moves only a small part of the body wall away from an object touching the skin, while the rest of the animal remains stationary. Amazingly, the precision of this localized behavioral response is similar to the spatial discrimination threshold of the human fingertip, although the leech skin is innervated by an order of magnitude fewer mechanoreceptors and each midbody ganglion contains only 400 individually identified neurons in total. Prior studies suggested that this behavior is controlled by a three-layered feed-forward network, consisting of four mechanoreceptors (P cells), approximately 20 interneurons and 10 individually characterized motor neurons, all of which encode tactile stimulus location by overlapping, symmetrical tuning curves. Additionally, encoding of mechanical force was attributed to three types of mechanoreceptors reacting to distinct intensity ranges: T cells for touch, P cells for pressure, and N cells for strong, noxious skin stimulation. In this study, we provide evidences that tactile stimulus encoding in the leech is more complex than previously thought. Combined electrophysiological, anatomical, and voltage sensitive dye approaches indicate that P and T cells both play a major role in tactile information processing resulting in local bending. Our results indicate that tactile encoding neither relies on distinct force intensity ranges of different cell types, nor location encoding is restricted to spike count tuning. Instead, we propose that P and T cells form a mixed type population, which simultaneously employs temporal response features and spike counts for multiplexed encoding of touch location and force intensity. This hypothesis is supported by our finding that previously identified local bend interneurons receive input from both P and T cells. Some of these interneurons seem to integrate mechanoreceptor inputs, while others appear to use temporal response cues, presumably acting as coincidence detectors. Further voltage sensitive dye studies can test these hypotheses how a tiny nervous system performs highly precise stimulus processing

Characterizing human vestibular sensory epithelia for experimental studies: new hair bundles on old tissue and implications for therapeutic interventions in ageing.

Balance disequilibrium is a significant contributor to falls in the elderly. The most common cause of balance dysfunction is loss of sensory cells from the vestibular sensory epithelia of the inner ear. However, inaccessibility of inner ear tissue in humans severely restricts possibilities for experimental manipulation to develop therapies to ameliorate this loss. We provide a structural and functional analysis of human vestibular sensory epithelia harvested at trans-labyrinthine surgery. We demonstrate the viability of the tissue and labeling with specific markers of hair cell function and of ion homeostasis in the epithelium. Samples obtained from the oldest patients revealed a significant loss of hair cells across the tissue surface, but we found immature hair bundles present in epithelia harvested from patients >60 years of age. These results suggest that the environment of the human vestibular sensory epithelium could be responsive to stimulation of developmental pathways to enhance hair cell regeneration, as has been demonstrated successfully in the vestibular organs of adult mice

Simultaneous assessment of CO2 sensitivity in the respiratory network and its neurons

This work was funded by the National Science Foundation IOS-1 022442

Distinct forms of synaptic inhibition and neuromodulation regulate calretinin positive neuron excitability in the spinal cord dorsal horn

The dorsal horn (DH) of the spinal cord contains a heterogenous population of neurons that process incoming sensory signals before information ascends to the brain. We have recently characterized calretinin-expressing (CR+) neurons in the DH and shown that they can be divided into excitatory and inhibitory subpopulations. The excitatory population receives high-frequency excitatory synaptic input and expresses delayed firing action potential discharge, whereas the inhibitory population receives weak excitatory drive and exhibits tonic or initial bursting discharge. Here, we characterize inhibitory synaptic input and neuromodulation in the two CR+ populations, in order to determine how each is regulated. We show that excitatory CR+ neurons receive mixed inhibition from GABAergic and glycinergic sources, whereas inhibitory CR+ neurons receive inhibition, which is dominated by glycine. Noradrenaline and serotonin produced robust outward currents in excitatory CR+ neurons, predicting an inhibitory action on these neurons, but neither neuromodulator produced a response in CR+ inhibitory neurons. In contrast, enkephalin (along with selective mu and delta opioid receptor agonists) produced outward currents in inhibitory CR+ neurons, consistent with an inhibitory action but did not affect the excitatory CR+ population. Our findings show that the pharmacology of inhibitory inputs and neuromodulator actions on CR+ cells, along with their excitatory inputs can define these two subpopulations further, and this could be exploited to modulate discrete aspects of sensory processing selectively in the DH

A method for the three-dimensional reconstruction of Neurobiotin(TM)-filled neurons and the location of their synaptic inputs

Here, we describe a robust method for mapping the number and type of neuro-chemically distinct synaptic inputs that a single reconstructed neuron receives. We have used individual hypoglossal motor neurons filled with Neurobiotin by semi-loose seal electroporation in thick brainstem slices. These filled motor neurons were then processed for excitatory and inhibitory synaptic inputs, using immunohistochemical-labeling procedures. For excitatory synapses, we used anti-VGLUT2 to locate glutamatergic pre-synaptic terminals and anti-PSD-95 to locate post-synaptic specializations on and within the surface of these filled motor neurons. For inhibitory synapses, we used anti-VGAT to locate GABAergic pre-synaptic terminals and anti-GABA-A receptor subunit a1 to locate the post-synaptic domain. The Neurobiotin-filled and immuno-labeled motor neuron was then processed for optical sectioning using confocal microscopy. The morphology of the motor neuron including its dendritic tree and the distribution of excitatory and inhibitory synapses were then determined by three-dimensional reconstruction using IMARIS software (Bitplane). Using surface rendering, fluorescence thresholding, and masking of unwanted immuno-labeling, tools found in IMARIS, we were able to obtain an accurate 3D structure of an individual neuron including the number and location of its glutamatergic and GABAergic synaptic inputs. The power of this method allows for a rapid morphological confirmation of the post-synaptic responses recorded by patch-clamp prior to Neurobiotin filling. Finally, we show that this method can be adapted to super-resolution microscopy techniques, which will enhance its applicability to the study of neural circuits at the level of synapses

Axonal projections of Renshaw cells in the thoracic spinal cord

Renshaw cells are widely distributed in all segments of the spinal cord, but detailed morphological studies of these cells and their axonal branching patterns have only been made for lumbosacral segments. For these, a characteristic distribution of terminals was reported, including extensive collateralization within 1-2 mm of the soma, but then more restricted collaterals given off at intervals from the funicular axon. Previous authors have suggested that the projections close to the soma serve inhibition of motoneurons (known to be greatest for the motor nuclei providing the Renshaw cell excitation) but that the distant projections serve mainly the inhibition of other neurons. However, in thoracic segments, inhibition of motoneurons is known to occur over two to three segments (20-40 mm) from the presumed somatic locations of the Renshaw cells. Here, we report the first detailed morphological study of Renshaw cell axons outside the lumbosacral segments, which investigated whether this different distribution of motoneuron inhibition is reflected in a different pattern of Renshaw cell terminations. Four Renshaw cells in T7 or T8 segments were intracellularly labeled with neurobiotin in anesthetized cats and their axons traced for distances ≥6 mm from the somata. The only morphological difference detected within this distance in comparison with Renshaw cells in the lumbosacral cord was a minimal taper in the funicular axons, where in the lumbosacral cord this is pronounced. Patterns of termination were virtually identical to those in the lumbosacral segments, so we conclude that these patterns are unrelated to the pattern of motoneuronal inhibition